Creatine kinase (CK) catalyzes the following reaction: EQU creatine phosphate+ADP.rarw..fwdarw.creatine+ATP
Measurement of CK activity in body fluids, especially in serum, is very important for the diagnosis of skeletal and myocardiac muscle disorder, especially, acute myocardial infarction (AMI). CK consists of two subunits, M (muscle type) and B (brain type), and three kinds of isoenzymes are present in cytoplasm, CK-MM, MB and BB. In particular, the separate measurement of CK-MB is recommended for specific diagnosis of myocardial infarction.
It has been elucidated that there are several post-translational isoforms of CK-MM and MB isoenzymes (George S., et al., J. Biol. Chem., 259, 2667-74 (1984). M subunit originally present in tissue, for example in cardiac muscle, is "tissue type M subunit (designated M.sub.T)." Once M.sub.T subunits in CK-MM or MB are released into blood stream, carboxypeptidase in plasma cleaves C-terminal lysine of the M.sub.T subunits, generating "serum type M subunit (designated M.sub.S)." Thus, CK-MM is converted from MM3 (tissue type, M.sub.T M.sub.T) via MM2 (hybrid type M.sub.T M.sub.S) to MM1 (serum type, M.sub.S M.sub.S).
It has been also clarified that the measurement of CK isoforms is a good index for earlier diagnosis of AMI and earlier detection of reperfusion after treatment of coronary thrombolysis. For example, it was reported that the ratio of MM3 to MM1 (MM3/MM1) is approximately 0.3 in healthy subjects, however, the ratio rises to 2.0, 3 to 6 hours after onset of AMI (Hashimoto H., et al., Circulation, 71, 366 (1985). Jaffe, et al. also reported (Circulation, 74, 105-9(1986) that 86% of the ratio in the first serum samples collected after onset from AMI patients exceeded its reference interval, on the other hand, the mean of total CK or CK-MB, those of which are conventional markers for diagnosis of AMI, still remained in their reference intervals.
Accordingly, the measurement of CK isoforms is very useful for the earlier diagnosis of AMI, compared with conventional AMI markers such as total CK activity and CK-MB. Earlier diagnosis of AMI makes it possible to treat AMI patients earlier with coronary thrombolysis or angioplasty.
The methods for the measurement of CK isoforms which have been reported are electrophoresis, isoelectric focusing, chromatofocusing, and liquid chromatography. Those methods are complicated, time-consuming, difficult to automate, and thus inadequate for emergency and routine assay.
Immunoassay is widely used in clinical chemistry field, and is a very excellent method because of its high sensitivity and specifity. Shah et al. showed immunoassay of CK isoforms using isoforms of specific antibodies (U.S. Pat. No. 4,900,662). This method, however, requires a long incubation time and separation and washing steps of solid phase from liquid phase, indicating that the method is complicated, laborious and unsuitable for emergency assay.
Immunoinhibition assay is also used in clinical laboratories. This method employs antibodies that inhibit part of enzyme activity such as particular isoenzymes. This method is being applied to the measurement of CK-MB isoenzyme using antibodies that inhibit CK-M (both M.sub.T and M.sub.S). In the assay, after all M subunit activity is inhibited by the antibodies, the remaining B subunit activity is then measured (Japanese Patent Publication 19239/1981 and 20274/1983). The immonoinhibition assay requires no separation procedures. The assay is completed in a short time and is easily automated, indicating that the assay is adequate for emergency and routine measurement. Immunoinhibition assay, however requires very specific antibodies. Antibodies used for immunoinhibition assay must have the ability not only to distinguish isoenzymes or isoforms from each other but also to inhibit particular enzyme activity. Generally, the antibodies against enzymes do not have either the distinguishing or inhibiting ability. The antibodies disclosed in the U.S. Pat. No. 4,900,662 cannot inhibit the enzymatic activity, meaning that they cannot be used in the immonoinhibition assay of CK isoform. The antibodies disclosed in the U.S. Pat. Nos. 19239 and 20274 cannot distinguish the CK-M.sub.T subunit from CK-M.sub.S. They also cannot be used in the immunoinhibition assay.
Therefore, a faster and easier method for the measurement of CK isoforms is eagerly required. The present invention provides the method for the measurement of CK isoform by immunoinhibition assay.